Schwann cells regulate tumor cells and cancer-associated fibroblasts in the pancreatic ductal adenocarcinoma microenvironment

Neuropathy is a feature more frequently observed in pancreatic ductal adenocarcinoma (PDAC) than other tumors. Schwann cells, the most prevalent cell type in peripheral nerves, migrate toward tumor cells and associate with poor prognosis in PDAC. To unveil the effects of Schwann cells on the neuro-stroma niche, here we perform single-cell RNA-sequencing and microarray-based spatial transcriptome analysis of PDAC tissues. Results suggest that Schwann cells may drive tumor cells and cancer-associated fibroblasts (CAFs) to more malignant subtypes: basal-like and inflammatory CAFs (iCAFs), respectively. Moreover, in vitro and in vivo assays demonstrate that Schwann cells enhance the proliferation and migration of PDAC cells via Midkine signaling and promote the switch of CAFs to iCAFs via interleukin-1α. Culture of tumor cells and CAFs with Schwann cells conditioned medium accelerates PDAC progression. Thus, we reveal that Schwann cells induce malignant subtypes of tumor cells and CAFs in the PDAC milieu.

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Hao Chen, Lingxi Jiang; Baiyong Shen 2023/06/30 scRNA seq data was performed using HiSeqXten (Illumina, San Diego, CA, USA) with a 150 bp bp paired-end run. Microarray-Based ST ST was sequenced using an an Illumina sequencer (Illumina) with a 150 bp bp paired-end run. Bulk RNA seq data was collected by by an an Illumina HiSeq X10 platform (2 (2 × 150 bp). The amplification signal of of qPCR was acquired by by qTOWER384G (Analytikjena). Tryptic peptides were analyzed using a Orbitrap Fusion LUMOS mass spectrometer (Thermo Fisher Scientific) coupled to to an an Easy-nLC 1200 via an an Easy Spray (Thermo Fisher Scientific). Luciferin emission imaging of of isoflurane-anesthetized animals was performed using the IVIS Spectrum (Tanon). Flow cytometry was performed using FACS (Becton-Dickinson, Bedford, MA, USA).
Statistical analyses were performed using the GraphPad Prism version 8.0. IHC/IF positive area and intensity were calculated by by QuPath Version 0.1.2 (https://qupath.github.io/). Patients in in the tissue microarray were divided into low and high SC SC area/iCAFs number groups according to to the optimal cut-off value calculated using the X-Tile software. K-M curve and Cox regression analysis was performed to to assess the association with overall survival using SPSS v23 (IBM Inc.).Migrated cells number was quantified in in an an automated fashion using the ImageJ v1.53e software. scRNA-seq and ST ST data analysis was performed by by NovelBio Bio-Pharm Technology Co., Ltd. with the NovelBrain Cloud Analysis Platform. Flow cytometry results were analyzed using the FlowJo V10 software. nature portfolio | reporting summary

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Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. We collected the human pancreatic cancer and adjacent normal tissue from 187 PDAC patients, including 112 males and 75 females with median ages of 62 and 67 years old. The univariate analysis in Figure 1e included the gender, and the result showed no significant difference. Thus, we didn't include sex or gender analysis in the following study.
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All the samples in this study were collected as biological replicates and the data are highly reproducible. scRNA-seq, ST were conducted as 4 biologically replications. Tissue microarrays contain 187 paired replications. Bulk RNA seq for tumor cells, cell growth assays, cell migration, cell invasion, flow cytometry, IF, IHC, qPCR assays, MS were conducted as 3 biological independent experiments (the exact number is mentioned in the respective figure legend). Western blotting, ELISA, bulk RNA seq for CAFs, were conducted as 2 biological independent experiments (the exact number is mentioned in the respective figure legend).
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Primary fibroblasts were confirmed by morphology and expression of CAF markers, including -SMA and FAP, by Western blotting and IF. All cell lines were authenticated by STR profiling.
All cell lines were tested negative using PCR Mycoplasma Detection Kit (G238, ABM) No commonly misidentified cell lines were used.
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Nude BALB/c mice (6 weeks old) were purchased from the Chinese Academy of Sciences (Shanghai, China) and maintained in a specific pathogen-free facility. Animals were housed in an negative pressured isolator under 12h light-dark cycles with temperature at 22 C and humidity set points 50-60%

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For the cell cycle assay, 1×106 cells were collected and fixed in 75% ethanol at 4°C overnight. DNA was stained using propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, USA), according to the manufacturer's instructions. For the apoptotic assay, 1×106 cells were incubated with 5 L of FITC-conjugated annexin V (BD Biosciences, 556419) and 5 l of PI (BD Biosciences, 550825) for 15 min at room temperature in the dark.
5,000 cells were acquired for each sample. No cell sorting was performed in this manuscript.
The FACS assay used in this study is an the established protocol for cell cycle and apoptotic assay analysis.